ORIGINAL RESEARCH
published: 31 July 2018
Edited by:
Fiona Walsh,
Maynooth University, Ireland
Reviewed by:
Xu Jia,
Chengde Medical College, China
Luca A. Vitali,
University of Camerino, Italy
*Correspondence:
Francesco Iannelli
francesco.iannelli@unisi.it
Gianni Pozzi
gianni.pozzi@unisi.it
† Present address:
Marco Cassone,
Division of Geriatric and Palliative Care
Medicine, University of Michigan,
Ann Arbor, MI, United States
Marco R. Oggioni,
Department of Genetics, University
of Leicester, Leicester,
United Kingdom
Gian Maria Rossolini,
Department of Experimental
and Clinical Medicine, University
of Florence, Florence, Italy
Specialty section:
This article was submitted to
Antimicrobials, Resistance
and Chemotherapy,
a section of the journal
Frontiers in Microbiology
Received: 22 February 2018
Accepted: 04 July 2018
Published: 31 July 2018
Citation:
Iannelli F, Santoro F, Santagati M,
Docquier J-D, Lazzeri E, Pastore G,
Cassone M, Oggioni MR,
Rossolini GM, Stefani S and Pozzi G
(2018) Type M Resistance
to Macrolides Is Due to a Two-Gene
Efflux Transport System of the
ATP-Binding Cassette (ABC)
Superfamily. Front. Microbiol. 9:1670.
doi: 10.3389/fmicb.2018.01670
Frontiers in Microbiology | www.frontiersin.org
doi: 10.3389/fmicb.2018.01670
Type M Resistance to Macrolides Is
Due to a Two-Gene Efflux Transport
System of the ATP-Binding Cassette
(ABC) Superfamily
Francesco Iannelli 1* , Francesco Santoro 1 , Maria Santagati 2 , Jean-Denis Docquier 1 ,
Elisa Lazzeri 1 , Gabiria Pastore 1 , Marco Cassone 1† , Marco R. Oggioni 1† ,
Gian M. Rossolini 1† , Stefania Stefani 2 and Gianni Pozzi 1*
1
Department of Medical Biotechnologies, University of Siena, Siena, Italy, 2 Section of Microbiology, Department
of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy
The mef(A) gene was originally identified as the resistance determinant responsible
for type M resistance to macrolides, a phenotype frequently found in clinical isolates
of Streptococcus pneumoniae and Streptococcus pyogenes. MefA was defined as a
secondary transporter of the major facilitator superfamily driven by proton-motive force.
However, when characterizing the mef(A)-carrying elements Tn1207.1 and 81207.3,
another macrolide resistance gene, msr(D), was found adjacent to mef(A). To define the
respective contribution of mef(A) and msr(D) to macrolide resistance, three isogenic
deletion mutants were constructed by transformation of a S. pneumoniae strain
carrying 81207.3: (i) 1mef(A)–1msr(D); (ii) 1mef(A)–msr(D); and (iii) mef(A)–1msr(D).
Susceptibility testing of mutants clearly showed that msr(D) is required for macrolide
resistance, while deletion of mef(A) produced only a twofold reduction in the minimal
inhibitory concentration (MIC) for erythromycin. The contribution of msr(D) to macrolide
resistance was also studied in S. pyogenes, which is the original host of 81207.3. Two
isogenic strains of S. pyogenes were constructed: (i) FR156, carrying 81207.3, and (ii)
FR155, carrying 81207.3/1msr(D). FR155 was susceptible to erythromycin, whereas
FR156 was resistant, with an MIC value of 8 µg/ml. Complementation experiments
showed that reintroduction of the msr(D) gene could restore macrolide resistance in
1msr(D) mutants. Radiolabeled erythromycin was retained by strains lacking msr(D),
while msr(D)-carrying strains showed erythromycin efflux. Deletion of mef(A) did not
affect erythromycin efflux. This data suggest that type M resistance to macrolides in
streptococci is due to an efflux transport system of the ATP-binding cassette (ABC)
superfamily, in which mef(A) encodes the transmembrane channel, and msr(D) the two
ATP-binding domains.
Keywords: ABC transporter, macrolide efflux, 81207.3, prophage, mef(A), msr(D), Streptococcus pneumoniae,
Streptococcus pyogenes
INTRODUCTION
Macrolides are antibiotic compounds composed of 14 (erythromycin and clarithromycin), 15
(azithromycin), or 16 (josamycin, spiramycin, tylosin)-membered lactones to which amino and/or
neutral sugars are linked (Roberts et al., 1999; Roberts, 2008). Resistance to macrolides in
streptococci is usually associated with two major mechanisms: (i) target site modification, arising
1 July 2018 | Volume 9 | Article 1670
Iannelli et al.
from the presence of erm(B) or erm(A) subclass erm(TR)
belonging to the class of erm (erythromycin ribosome
methylation) methylase genes (Leclercq and Courvalin, 1991;
Seppala et al., 1998) and (ii) drug efflux, associated to the
presence of mef (A) (macrolide efflux) genes (Clancy et al., 1996;
Sutcliffe et al., 1996; Tait-Kamradt et al., 1997). Methylation of
23S rRNA causes a reduced binding to macrolide, lincosamide,
and streptogramin B antibiotics (MLSB phenotype), whereas
active efflux of macrolides confers a low level of resistance to
resistance only to 14- and 15-membered macrolides, but not to
16-membered macrolides, lincosamides, and streptogramin B
antibiotics (M phenotype; Weisblum, 1995; Roberts et al., 1999).
The macrolide efflux mef (A) gene was originally described
in Streptococcus pyogenes (Clancy et al., 1996) while the allelic
variant mef (E) was first described in Streptococcus pneumoniae
(Tait-Kamradt et al., 1997). These variants are highly homologous
(about 90% nucleotide identity) and are grouped in the same
mef (A) class of macrolide resistance genes (Roberts et al.,
1999). The mef (A) gene was not found only in S. pyogenes and
S. pneumoniae, but also in a wide variety of other streptococcal
species such as Streptococcus agalactiae, Streptococcus mitis,
Streptococcus oralis, and Streptococcus salivarius, in other
Gram-positive genera including Corynebacterium, Enterococcus,
Micrococcus, and Staphylococcus, and in Gram-negative genera
such as Acinetobacter, Bacteroides, and Neisseria (Roberts et al.,
1999; Klaassen and Mouton, 2005; Santagati et al., 2009). In
some countries, the mef (A) gene has become more common
than erm(B) in macrolide-resistant S. pneumoniae and S. pyogenes
(Green et al., 2006; Reinert et al., 2006; Rudolph et al., 2013)
and it has been used as a marker for molecular epidemiology
studies (Daly et al., 2004; Klaassen and Mouton, 2005; Amezaga
and McKenzie, 2006). The mef (A) allelic variants are carried
by different genetic elements. In S. pneumoniae, we described
a 7244-bp non-conjugative element named Tn1207.1 carrying
mef (A), whereas mef (E) was found to be carried by the 5532-
bp pneumococcal genetic element (mega; Santagati et al., 2000;
Gay and Stephens, 2001; Del Grosso et al., 2002, 2006). In
S. pyogenes, mef (A) is carried by 81207.3, a 52,491-bp prophage
which we found in the erythromycin-resistant clinical strain
2812A, transferable to a variety of streptococcal species and
whose left 7244-bp sequence is 100% identical to pneumococcal
Tn1207.1 (Santagati et al., 2003; Pozzi et al., 2004; Iannelli et al.,
2014a). Other mef (A)-carrying prophages in clinical isolates of
S. pyogenes include 810394.4 (Banks et al., 2003), 8m46.1, and
its variant VP_00501.1 (Brenciani et al., 2004, 2010; Vitali et al.,
2016).
At first, Mef(A) was defined as a secondary transporter of the
major facilitator superfamily (MFS)1 and considered to be the
only gene product responsible for type M macrolide resistance, as
shown by cloning and functional characterization in Escherichia
coli (Clancy et al., 1996). However, in mef (A)-carrying genetic
elements such as Tn1207.1 and 81207.3, another macrolide
resistance gene, msr(D), was always found adjacent to mef (A),
and msr(D) was shown to contribute to the macrolide efflux
resistance of streptococcal strains carrying the mef (A)–msr(D)
1
http://www.tcdb.org/
Frontiers in Microbiology | www.frontiersin.org
ABC-Cassette Responsible for Macrolide Efflux
tandem pair (Iannelli et al., 2004; Ambrose et al., 2005; Vitali
et al., 2016; Zhang et al., 2016; Tatsuno et al., 2018).
To assess the relative contribution of mef (A) and msr(D)
to macrolide efflux, we generated in-frame isogenic mutants
of S. pneumoniae and S. pyogenes carrying mef (A)-msr(D) to
be used in functional studies. Mutant strains were tested by
(i) determining the minimal inhibitory concentration (MIC)
of erythromycin and (ii) assaying the actual intracellular
accumulation of radiolabeled erythromycin. Results of functional
studies were in accordance with bioinformatics analysis
predicting that the tandem mef (A)-msr(D) gene pair encodes
an efflux transport system of the ATP-binding cassette (ABC)
superfamily.
MATERIALS AND METHODS
Bioinformatic Softwares
Protein sequence analysis was performed with the softwares
Phyre2 (Kelley et al., 2015) and TMpred. Conserved
aminoacids were identified with the PSI-BLAST multiple
sequence alignment. Terminator sequence was predicted with
RNAstructure ver. 5.02 (Mathews Lab). Nucleotide sequence
analysis was performed using Microbial Nucleotide BLAST with
the Megablast algorithm2 .
Bacterial Strains and Growth Conditions
Streptococcal strains used in this work and their relevant
properties are reported in Table 1. Bacteria were routinely
grown in Tryptic Soy Broth (TSB) or Tryptic Soy Agar
(Difco) supplemented with 3% horse blood at 37◦ C. When
required, 500 µg/ml streptomycin, 10 µg/ml novobiocin,
0.5 µg/ml erythromycin, 100 µg/ml spectinomycin, and 3 µg/ml
chloramphenicol were added to both liquid and solid media.
Construction of the Isogenic
S. pneumoniae Mutant Strains
To allow construction of mutants deleted for the macrolide
resistance genes object of this study, and to facilitate further
analysis, it was essential to work in a S. pneumoniae strain
which is readily transformable and whose genomic sequence is
available (Hoskins et al., 2001). For this reason, we transferred
81207.3 to the chromosome of unencapsulated laboratory strains
of S. pneumoniae which are derived from type 2 strain D39
(Iannelli et al., 1999; Pearce et al., 2002).
Integration of Tn1207.1 or 81207.3 into the chromosome of
S. pneumoniae occurs within the cds of celB, a gene involved
in DNA uptake during transformation, and disruption of celB
leads to impairment of competence for genetic transformation in
S. pneumoniae strains carrying Tn1207.1 or 81207.3 (Santagati
et al., 2000, 2003). For this reason, strain FR125 was constructed
in which celB was deleted and integration of 81207.3 after
mating occurred at a different chromosomal location (Iannelli
and Pozzi, unpublished results). Subsequently, strain FR183, a
2
https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE~=~BlastSearch&BLAST_
SPEC~=~MicrobialGenomes
2 July 2018 | Volume 9 | Article 1670
Iannelli et al. ABC-Cassette Responsible for Macrolide Efflux

TABLE 1 | Bacterial strains.

Strain Relevant propertiesa Origin (references)

S. pneumoniae
D39 Type 2 Avery’s strain Pearce et al., 2002; Lanie et al., 2007
Rx1 Unencapsulated derivative of D39 Smith and Guild, 1979; Pearce et al., 2002
DP1004 Unencapsulated derivative of D39; str-41; SmR Salles et al., 1992; Iannelli and Pozzi, 2004
FP11 Rx1 unencapsulated competence deficient derivative, 1comC; NovR , CmR Iannelli and Pozzi, 2007; Santoro et al., 2010
FR122 FP11 derivative carrying 81207.3; NovR , CmR , EmR This study
FR125 Rx1 unencapsulated competence deficient derivative carrying 81207.3; 1comC, Iannelli and Pozzi, unpublished results
str-41, 1celB; CmR , SmR , SpcR , EmR
FR183 DP1004 derivative carrying 81207.3 (by transformation with FR125 chromosomal This study
DNA); SmR , EmR
FP39 FR183 derivative, 1mef(A) and 1msr(D); SmR , CmR This study
FP40 FR183 derivative, 1mef(A); SmR , EmR , CmR This study
FP59 FR183 derivative, 1msr(D); SmR , SpcR This study
FP92 FP59 derivative, msr(D) (by transformation with a msr(D)-containing PCR fragment This study
amplified from FR183); SmR , EmR
FR139 FP11 derivative carrying 81207.3 – 1msr(D) (by conjugation with FP59); NovR , SpcR This study
S. pyogenes
D471 Streptomycin-resistant serotype M6 strain from RU collection, SmR Scott and Fischetti, 1983
FR156 D471 derivative carrying 81207.3 (by conjugation with FR122); SmR , EmR This study
FR155 D471 derivative carrying 81207.3 – 1msr(D) (by conjugation with FR139); SmR , SpcR This study
FR160 FR155 derivative carrying 81207.3 (by conjugation with FR122); SmR , EmR , SpcR This study
a nov-1 and str-41 indicate point mutations conferring resistance to novobiocin and streptomycin, respectively. Nov, novobiocin; Sm, streptomycin; Cm, chloramphenicol;
Tc, tetracycline; Em, erythromycin; Spc, spectinomycin; Km, kanamycin.

transformable S. pneumoniae DP1004 derivative strain carrying
81207.3 integrated elsewhere than celB, was obtained by
transformation with the chromosomal DNA purified from FR125
(Table 1).
Matings
Mating experiments were performed as already described
(Iannelli and Pozzi, 2007). Briefly, cells were grown separately in
presence of appropriate antibiotics until the end of exponential
phase (OD590nm = 0.8). Cells were mixed at 1:10 ratio
(Donor/Recipient), harvested by centrifugation and plated.
Following 4-h incubation, cells were harvested by scraping
the plates. Selection of recombinants was carried out with
a multilayer plating procedure. Transfer frequencies were
determined by plating alone each parent strain that was also
checked for spontaneous antibiotic resistance acquisition.
Oligonucleotide Primers
Oligonucleotide primers used for mutagenesis, sequencing, and
PCR selection of recombinant strains are listed in Supplementary
Table S1.
PCR Mutagenesis
Gene splicing by overlap extension (gene SOEing) was used to
generate the mutagenic constructs for gene deletions as already
described (Pearce et al., 2002; Iannelli and Pozzi, 2004). The
deletion of mef (A) and msr(D) genes was obtained with a
mutagenic construct containing the ami/cat cassette flanked
at the left by a 195-bp DNA fragment corresponding to
nucleotides 3112–3307 of 81207.3 and at the right by a fragment
corresponding to the 532 nucleotides located downstream of
the msr(D) stop codon. The ami/cat chloramphenicol-resistance
cassette was obtained with the primer pair IF38/IF39 using the
E. coli pEVP3 plasmid as template (Claverys et al., 1995). The
flanking regions were amplified, respectively, with the primer
pairs IF105/F35 and IF183/IF175 from chromosomal DNA of
FR183. To minimize possible polar effects, the mef (A) coding
sequence (cds) was deleted in-frame by allelic replacement with
the cds of the cat chloramphenicol-resistance gene. The cat cds
was amplified with primer pair IF184/IF39 from a cat(pC194)-
carrying strain (Iannelli et al., 2014b) and flanked by a 1116-
bp DNA region located upstream mef (A) start codon and a
DNA fragment spanning the 449 nucleotides downstream of
the mef (A) stop codon, obtained, respectively, with the primer
pairs IF176/IF182 and IF185/F20 from FR183 chromosome.
A genetic construct containing the ami/spc cassette flanked by
the 281-bp and 532-bp DNA fragments located respectively
upstream and downstream of msr(D) cds was produced to
inactivate msr(D). The primer couple IF100/IF101 was used to
amplify the spectinomycin-resistance cassette from the pR412
plasmid (Martin et al., 2000). This cassette was flanked by
the msr(D) flanking segments amplified with the primer pair
IF171/IF174 from FR183 chromosome. Linear PCR constructs
were used directly as donor DNA in transformation experiments.
Mutant strains were selected for acquisition of chloramphenicol
or spectinomycin resistance and the desired mutations were
confirmed by sequencing. PCR-based selection of S. pneumoniae
complemented strains was carried out with the primers pair
IF169/IF170 according to the protocol already described (Iannelli
and Pozzi, 2004).

Frontiers in Microbiology | www.frontiersin.org 3 July 2018 | Volume 9 | Article 1670

Iannelli et al.
Erythromycin Sensitivity Determination
The MIC was assessed by microdilution techniques as suggested
by the (Clinical and Laboratory Standards Institute [CLSI],
2017). Briefly, MICs were determined as follows: bacterial cells
were grown until exponential growth phase (OD590nm = 0.25)
in TSB medium and stored at −70◦ C in 10% glycerol, then
frozen cultures were thawed and diluted at 5 × 104 CFU/ml
in TSB broth containing serial twofold dilutions of antibiotic,
and incubated at 37◦ C for 18 h. Bacterial growth was
determined turbimetrically using the microplate ELISA reader
VERSAmax (Molecular Devices). Results are presented as the
geometric mean and are derived from at least three experiments.
S. pneumoniae ATCC49619 was used as reference control strain
as recommended (Clinical and Laboratory Standards Institute
[CLSI], 2017).
Erythromycin Efflux Assay
Frozen starter culture was diluted 100-fold in 100 ml warm
TSB medium and grown at 37◦ C until an optical density at
590 nm of 0.25. Radiolabeled erythromycin [N-methyl-14 C]
(50 µCi/mmol, purchased from Bio Trend, Germany) was added
at a final concentration of 0.2 µg/ml (Sutcliffe et al., 1996).
Erythromycin uptake was assessed by taking 5-ml samples from
each culture every 10 min for 40 min after the addition of
[14 C]erythromycin, filtering samples with prewet GF/C glass
microfiber filters (Whatman), and washing the filters twice with
5 ml of a 0.9% NaCl and 1 µg/ml erythromycin solution (Sutcliffe
et al., 1996). Filters were dried O.N., dissolved in 10 ml of
Insta-Gel Plus liquid (Packard) and the [14 C] erythromycin
cell-associated was counted with a TRI-CARB 2000 CA Liquid
Scintillation Analyzer (Packard).
RESULTS
mef(A) and msr(D) Encode an ABC
Transport System
In clinical isolates of macrolide resistant streptococci, the
macrolide efflux gene, mef (A), is found in tandem with
msr(D), a gene which encodes for an ATP-binding transporter
(Santagati et al., 2000, 2003; Gay and Stephens, 2001; Banks
et al., 2003; Brenciani et al., 2004). Bioinformatic analysis
of the prototypic tandem mef (A)-msr(D) from Tn1207.1
(Santagati et al., 2000) and their flanking regions revealed
that they constitute a two-gene operon which is 3290 bp long,
with only one putative promoter sequence −35 TTGCTT,
extended −10 TGTGTTAAAAT (nucleotides 2890–2895
and 2908–2918, respectively, Genbank AF227520) located
upstream of the mef (A) start codon and a single predicted
terminator sequence (nucleotides 6143–6179, Genbank
AF227520) located downstream of the msr(D) stop codon.
As previously demonstrated (Ambrose et al., 2005; Chancey
et al., 2015), RT-qPCR analysis confirmed that mef (A) and
msr(D) genes were part of a single transcriptional unit (data
not shown). Sequence analysis of the Mef(A) protein with
TMpred showed the presence of 12 predicted transmembrane
Frontiers in Microbiology | www.frontiersin.org
ABC-Cassette Responsible for Macrolide Efflux
domains, which have the potential to form six transmembrane
helices (Figure 1).When the protein sequence of Msr(D) was
analyzed by the PSI-BLAST multiple sequence alignment, it was
possible to show the presence of two nucleotide binding domains
(NBDs) typical of ATP-binding transporters (spanning amino
acids 12–154, and 304–463, Genbank AAG12999.1), and a long
predicted alpha helical structure (spanning amino acids 181–
227) between the two NBDs (Figure 1). Altogether, mef (A) and
msr(D) appear to constitute a two-gene efflux transport system of
the ABC superfamily, mef (A) encodes the pore (transmembrane
channel), and msr(D) the two ATP-binding domains.
mef(A) and msr(D) Are Found in Tandem
in Bacterial Genomes
The mef (A) gene sequence (GenBank AF227520) was used as a
query to interrogate the database of 20,187 complete Microbial
genomes (accessed in May 2018). The mef (A) gene was found
in 37 genomes, of those: 29 were streptococcal genomes and
eight belonged to other genera (namely Turicibacter, Clostridium,
Enterococcus, Clostridioides, Gardnerella, and Bacillus). In 33 out
of 37 cases, the mef (A) gene was in tandem with msr(D), while
in the remaining four cases, msr(D) was substituted by a gene
coding for a putative nucleotidyltransferase. No information on
macrolide resistance was available for these four genome strains.
Type M Macrolide Resistance Depends
on the ATP-Binding Transporter Msr(D)
To define the respective contribution of mef (A) and msr(D)
gene products to macrolide efflux resistance, isogenic deletion
FIGURE 1 | The mef(A)–msr(D) operon is 3290-bp-long and includes a single
promoter sequence (P) and a terminator sequence (T). Mef(A) contains 12
transmembrane domains spanning the whole protein (red bars). Msr(D)
contains two nucleotide binding domains (NBDs, blue boxes) connected by
an alpha helical structure (green cylinder). Each NBD has seven motifs (blue
bars): (1) the “A-loop” that provides an aromatic side chain residue that
interacts with the adenine ring of bound nucleotide (phenylalanine in both
NBDs); (2) the “Q-loop” involved in the interaction with the transmembrane
domain and with the γ-phosphate through a water bond; (3) the “P-loop” (or
“Walker-A” motif) that binds the nucleotide; (4) the “LSGGQ motif” (also called
“C-loop” or “ABC signature motif”) which contacts the nucleotide in the
ATP-bound state; (5) the “Walker-B” motif that has a conserved glutamate
residue that orchestrates the nucleophilic attack on ATP via a water molecule;
(6) the “D-loop” involved in the contact between the two NBDs through an
interaction with the Walker-A asparagine residue; and (7) the “switch motif”
that contains a histidine side chain thought to contribute to the catalytic
reaction. Both C-loop motifs of Msr(D), MSGGE and LSGGE, are not identical
to the consensus (LSGGQ).
4 July 2018 | Volume 9 | Article 1670
Iannelli et al. ABC-Cassette Responsible for Macrolide Efflux

mutants were constructed in S. pneumoniae for the mef (A)– TABLE 2 | Sensitivity to erythromycin.
msr(D) tandem pair carried by two different genetic elements
Strain Genotype MIC of erytromycin (µg/ml)a Phenotype
(Figure 2). Three isogenic deletion mutants were constructed
in the 81207.3-carrying strain FR183: (i) FP39, 1mef (A)– Streptococcus pneumoniae
1msr(D); (ii) FP40, 1mef (A); and (iii) FP59, 1msr(D). In FR183 mef(A), msr(D) 8 Resistant
FP39, a 2748-bp DNA fragment (position 3308–6055, GenBank FP39 1mef(A), 1msr(D) 0.06 Sensitive
AY657002) containing mef (A) and msr(D) was deleted and FP40 1mef(A), msr(D) 4 Resistant
replaced with the 850-bp ami/cat cassette. In FP40, a 1218- FP59 mef(A), 1msr(D) 0.12 Sensitive
bp DNA fragment (position 3255–4472, GenBank AY657002) DP1004b – 0.03 Sensitive
containing the cds of mef (A) was deleted and replaced in-frame Streptococcus pyogenes
with the 651-bp cds of the cat gene. In FP59, a 1464-bp DNA FR156 mef(A), msr(D) 8 Resistant
fragment (position 4592–6055, GenBank AY657002) containing FR155 mef(A), 1msr(D) 0.12 Sensitive
the msr(D) cds was deleted and replaced with the 894-bp ami/spc D471b – 0.03 Sensitive
cassette (Figure 2 and Table 1). a MIC interpretative standard: sensitive ≤ 0.25 µg/ml, intermediate = 0.5 µg/ml, and
Sensitivity to erythromycin of the S. pneumoniae strains resistant ≥ 1 µg/ml.
b DP1004 and D471 are parental erythromycin sensitive strains used as controls.
carrying mef (A)–msr(D) and of their isogenic mutants was tested
in liquid medium. It was clearly shown that msr(D) is required for
macrolide resistance (Figure 1 and Table 2). Deletion of mef (A) variants of mef (A) and msr(D) originally found in a macrolide-
produced only a twofold reduction of the MIC, while deletion resistant clinical strain of S. pneumoniae formerly described as a
of msr(D) produced a 64-fold MIC decrease (8 to 0.12 µg/ml), “mef (E)-positive isolate” (GenBank AF376746) (Del Grosso et al.,
and when both genes were deleted, the MIC decreased 128-folds 2002, 2004). Deletion mutants were constructed and tested for
(Table 2). Identical results were also obtained using the allelic erythromycin resistance. Results on MIC reduction were identical

FIGURE 2 | The mef(A)-msr(D) operon, carried by 81207.3, confers type M resistance to macrolides. Isogenic mutants were produced in a 81207.3-carrying
S. pneumoniae strain. CDSs were deleted by allelic replacement with mutagenic antibiotic resistance cassettes. In order to minimize polar effect, mef(A) CDS was in
frame deleted with the chloramphenicol resistance cat CDS. The genotype and relative erythromycin resistance phenotype of the pneumococcal isogenic strains are
schematized. The genes are reported as arrows while dotted lines indicate gene deletions; the transcriptional promoter (P) and the putative transcriptional terminator
(T) of the operon are reported.

Frontiers in Microbiology | www.frontiersin.org 5 July 2018 | Volume 9 | Article 1670

Iannelli et al. ABC-Cassette Responsible for Macrolide Efflux

to those observed for the mef (A)–msr(D) alleles described above
(data not shown).
Complementation of the msr(D) Deletion
To confirm the role of msr(D) in determining type M
resistance to macrolides, deletion of msr(D) was complemented
in S. pneumoniae FP59 carrying 81207.3/1msr(D). Competent
cells of FP59 were transformed with a 2206-bp PCR fragment
containing msr(D) obtained from wild type 81207.3, using
primer pair IF171/IF175. Transformants were selected by PCR
for the presence of the msr(D) gene, as previously described
(Iannelli and Pozzi, 2004). A total of four transformants
were selected out of 186 tested. All four showed acquisition
of erythromycin resistance, with an MIC value of 8 µg/ml.
A representative transformant was named FP92, checked by DNA
sequencing and used as a control in further experiments.
The msr(D) Gene in Streptococcus
pyogenes
The contribution of msr(D) to macrolide resistance was also
studied in S. pyogenes, which is the original host of 81207.3
(Santagati et al., 2003). Two isogenic strains of S. pyogenes
were constructed: (i) FR156, carrying 81207.3, and (ii) FR155,
carrying 81207.3/1msr(D) (Table 1). 81207.3 transfer from
S. pneumoniae donors to S. pyogenes recipients occurred at
frequencies of 10−7 recombinants per donor. FR155 lacking
msr(D) was susceptible to erythromycin (MIC of 0.12 µg/ml),
whereas FR156 was resistant, with an MIC of 8 µg/ml (Table 2).
These data show that in S. pyogenes the presence of the mef (A)
gene alone determines a fourfold increase of the MIC for
erythromycin, while when both mef (A) and msr(D) are present,
the MIC is increased by 256-folds (Table 2).
The 81207.3/1msr(D) S. pyogenes FR155 was complemented
in trans transferring a wild type 81207.3 element from
FR122. Recombinants were selected for erythromycin resistance
acquisition and in a representative recombinant, FR160, the
presence of both the wild type and mutated form of 81207.3 was
confirmed by DNA sequencing. FR160 showed an erythromycin
MIC value of 8 µg/ml.
Erythromycin Efflux
The function of mef (A) and msr(D) was studied by
testing incorporation of radiolabeled erythromycin in the
deletion mutants (Figure 3). Radiolabeled erythromycin
was retained/incorporated by strains lacking msr(D), while
msr(D)-carrying strains showed erythromycin efflux. This was
observed both in S. pneumoniae (Figure 3A) and in S. pyogenes
(Figure 3B). Accumulation of radiolabeled erythromycin was
maximum in the 1mef (A)–1msr(D) S. pneumoniae mutant.
The deletion of mef (A) did not affect erythromycin efflux:
1mef (A) mutant showed an erythromycin accumulation
comparable to that of the wild type strain, even if the kinetics of
accumulation were different (Figure 3A). Both the S. pneumoniae
and the S. pyogenes1msr(D) mutants showed an intracellular
accumulation of erythromycin which peaked already 10 min
after erythromycin addition (Figures 3A,B). These results
were confirmed in a series of control efflux assays which
included (i) testing deletion mutants derived a “mef (E)-positive
strain” carrying mef(A)–msr(D) allelic variants and (ii) testing
complemented strain FP92 (data not shown). Taken together,

FIGURE 3 | Efflux assay in S. pneumoniae (A) and S. pyogenes (B) isogenic mutant strains. The 1mef(A)–1msr(D) S. pneumoniae mutant showed the maximum
accumulation of radiolabeled erythromycin. Erythromycin efflux was not affected by the deletion of mef(A). S. pneumoniae and S. pyogenes1msr(D) mutants
showed an intracellular accumulation of erythromycin peaking 10 min after erythromycin addition. Genotype is indicated on the right of each graph. Cell-associated
erythromycin is reported as counts per minute (CPM). Arrows indicate the time point at which radiolabeled erythromycin was added. Full circles indicate the wild type
strain carrying mef(A) and msr(D), open circles the mef(A) and msr(D) deletion mutant, open diamonds the msr(D) deletion mutant, and open squares the mef(A)
deletion mutant. In all curves, the CPM baseline value at time 0 was subtracted from the CPM values of each time point.

Frontiers in Microbiology | www.frontiersin.org 6 July 2018 | Volume 9 | Article 1670

Iannelli et al.
these results suggest that type M resistance to macrolides
in streptococci is due to an efflux transport system of the
ABC superfamily, in which mef (A) encodes the transmembrane
channel, and msr(D) the two ATP-binding domains.
DISCUSSION
In this work, protein sequence analysis showed that Mef(A)
contains 12 transmembrane segments and Msr(D) contains two
ATP-binding domains (ABC domains). Analysis of bacterial
genomes showed that mef (A) and msr(D) are found in tandem
in 33 out of 37 cases. We constructed isogenic deletion
mutants in S. pneumoniae and S. pyogenes to define the
respective contribution of mef (A) and msr(D) to macrolide
resistance. Physiological studies by MIC determinations
and efflux assays, carried out in the original streptococcal
hosts, showed that msr(D) is essential for erythromycin
resistance and drug efflux, whereas mef (A) deletion produces
a twofold reduction of MIC and does not affect efflux.
Thus, it appears that mef (A) and msr(D) encode an ABC
transporter involved in macrolide efflux with Mef(A) as the
transmembrane channel, and Msr(D) as the cytoplasmatic
ATP-binding protein. ABC transporters are multidomain
membrane proteins minimally composed of two transmembrane
domains and two ATP-binding domains. Transmembrane
domains are responsible for binding and transport, while ATP-
binding domains for coupling the energy of ATP hydrolysis to
conformational changes in the transmembrane domains. These
four domains may belong to a single protein or to different
proteins.
In E. coli, the majority of transporters are secondary
transporters and rely on proton-motive force as a source of
energy3 . Streptococci are fermentative organisms that use ATP
as primary source of energy; accordingly, the majority of
transporters are ATP-dependent. Because of these metabolic
differences, it is likely that Mef(A) works as a major facilitator
when expressed in E. coli (Clancy et al., 1996), while in the
original streptococcal host, Mef(A) is coupled to the cognate
ATP-binding protein Msr(D) to function as an efflux system.
In this work, we show that the msr(D) gene (i) is always
in tandem with mef (A) (Figure 1), constituting a single
transcriptional unit, and (ii) confers type M resistance to
3
http://www.membranetransport.org/transportDB2
REFERENCES
Ambrose, K. D., Nisbet, A. D., and Stephens, D. S. (2005). Macrolide efflux in
Streptococcus pneumoniae is mediated by a dual efflux pump (mel and mef)
and is erythromycin inducible. Antimicrob. Agents Chemother. 49, 4203–4209.
doi: 10.1128/AAC.49.10.4203-4209.2005
Amezaga, M. R., and McKenzie, H. (2006). Molecular epidemiology of macrolide
resistance in beta-haemolytic streptococci of lancefield groups A, B, C and
G and evidence for a new mef element in group G streptococci that carries
allelic variants of mef and msr(D). J. Antimicrob. Chemother. 57, 443–449.
doi: 10.1093/jac/dki490
Banks, D. J., Porcella, S. F., Barbian, K. D., Martin, J. M., and Musser, J. M. (2003).
Structure and distribution of an unusual chimeric genetic element encoding
Frontiers in Microbiology | www.frontiersin.org
ABC-Cassette Responsible for Macrolide Efflux
macrolides by an ATP-dependent efflux mechanism. The msr(D)
gene product, Msr(D), is phylogenetically classified in the ABC-F
family of ABC transporters together with other ATP-binding
proteins, such as Vga and Lsa, which confer resistance also
to other ribosome targeting antibiotics such as lincosamides,
streptogramins, pleuromutilins, and ketolides. Proteins in this
family share a general architecture with two NBDs and a long,
mostly alpha helical, linker sequence connecting them. The
spectrum of antibacterial resistances conferred is variable among
members of this family, with some proteins giving resistance to
multiple classes of antibiotics (Lenart et al., 2015; Sharkey et al.,
2016).
Since deletion of mef (A) did not affect erythromycin efflux
and produced only a twofold reduction on erythromycin
MIC, it is possible that in the absence of Mef(A), Msr(D)
uses an alternative transmembrane protein to pump the
antibiotic out of the cell. A BlastP search in the genomes of
S. pneumoniae TIGR4 and S. pyogenes M1 revealed that there
are, respectively, three and one hypothetical transmembrane
proteins homologous to Mef(A). Site-specific mutagenesis of the
mef (A) homologous genes will be a first step for the identification
of alternative Msr(D) cognate transmembrane domains which
could complement the Mef(A) function.
AUTHOR CONTRIBUTIONS
FI and GPo designed the experiments. FI, FS, MS, J-DD, EL, GPa,
and MC performed the experimental work. FI, FS, MO, GR, SS,
and GPo analyzed and interpreted the data. FI, FS, and GPo wrote
the paper.
FUNDING
This study was supported by the European Commission grants
ANTIRESDEV HEALTH-F3-2009-241446 and Italian Ministry
of University and Research, project PRIN 2012 (2012WJSX8K).
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmicb.
2018.01670/full#supplementary-material
macrolide resistance in phenotypically diverse clones of group A Streptococcus.
J. Infect. Dis. 188, 1898–1908. doi: 10.1086/379897
Brenciani, A., Bacciaglia, A., Vignaroli, C., Pugnaloni, A., Varaldo, P. E., and
Giovanetti, E. (2010). Fm46.1, the main Streptococcus pyogenes element
carrying mef(A) and tet(O) genes. Antimicrob. Agents Chemother. 54, 221–229.
doi: 10.1128/AAC.00499-09
Brenciani, A., Ojo, K. K., Monachetti, A., Menzo, S., Roberts, M. C., Varaldo,
P. E., et al. (2004). Distribution and molecular analysis of mef(A)-containing
elements in tetracycline-susceptible and -resistant Streptococcus pyogenes
clinical isolates with efflux-mediated erythromycin resistance. J. Antimicrob.
Chemother. 54, 991–998. doi: 10.1093/jac/dkh481
Chancey, S. T., Bai, X., Kumar, N., Drabek, E. F., Daugherty, S. C., Colon, T.,
et al. (2015). Transcriptional attenuation controls macrolide inducible efflux
7 July 2018 | Volume 9 | Article 1670
Iannelli et al.
and resistance in Streptococcus pneumoniae and in other gram-positive bacteria
containing mef/mel(msr(D)) elements. PLoS One 10:e0116254. doi: 10.1371/
journal.pone.0116254
Clancy, J., Petitpas, J., Dib-Hajj, F., Yuan, W., Cronan, M., and Kamath, A. V., et al.
(1996). Molecular cloning and fuctional analysis of a novel macrolide-resistance
determinant, mefA, from Streptococcus pyogenes. Mol. Microbiol. 22, 867–879.
doi: 10.1046/j.1365-2958.1996.01521.x
Claverys, J. P., Dintilhac, A., Pestova, E. V., Martin, B., and Morrison, D. A.
(1995). Construction and evaluation of new drug-resistance cassettes
for gene disruption mutagenesis in Streptococcus pneumoniae, using
an ami test platform. Gene 164, 123–128. doi: 10.1016/0378-1119(95)
00485-O
Clinical and Laboratory Standards Institute [CLSI] (2017). Performance Standards
for Antimicrobial Susceptibility Testing, 27th Edn, Wayne, PA: CLSI supplement
Clinical and Laboratory Standards Institute.
Daly, M. M., Doktor, S., Flamm, R., and Shortridge, D. (2004). Characterization
and prevalence of mefA, mefE, and the associated mrs(D) gene in Streptococcus
pneumoniae clinical isolates. J. Clin. Microbiol. 42, 3570–3574. doi: 10.1128/
JCM.42.8.3570-3574.2004
Del Grosso, M., Camilli, R., Iannelli, F., Pozzi, G., and Pantosti, A. (2006).
The mef(E)-carrying genetic element (mega) of Streptococcus pneumoniae:
insertion sites and association with other genetic elements. Antimicrob. Agents
Chemother. 50, 3361–3366. doi: 10.1128/AAC.00277-06
Del Grosso, M., Iannelli, F., Messina, C., Santagati, M., Petrosillo, N., and Stefani, S.
et al. (2002). Macrolide efflux genes mef(A) and mef(E) are carried by different
genetic elements in Streptococcus pneumoniae. J. Clin. Microbiol. 50, 1230–1240.
doi: 10.1128/JCM.40.3.774-778.2002
Del Grosso, M., Scotto d’Abusco, A., Iannelli, F., Pozzi, G., and Pantosti, A. (2004).
Tn2009, a Tn916-like element containing mef(E) in Streptococcus pneumoniae.
Antimicrob. Agents Chemother. 48, 2037–2042. doi: 10.1128/AAC.48.6.2037-
2042.2004
Gay, K., and Stephens, D. S. (2001). Structure and dissemination of a chromosomal
insertion element encoding macrolide efflux in Streptococcus pneumoniae.
J. Infect. Dis. 184, 56–65. doi: 10.1086/321001
Green, M. D., Beall, B., Marcon, M. J., Allen, C. H., Bradley, J. S., Dashefsky, B., et al.
(2006). Multicentre surveillance of the prevalence and molecular epidemiology
of macrolide resistance among pharyngeal isolates of group A streptococci
in the USA. J. Antimicrob. Chemother. 57, 1240–1243. doi: 10.1093/jac/
dkl101
Hoskins, J., Alborn, W. E., Arnold, J., Blaszczak, L. C., Burgett, S., Dehoff,
B. S., et al. (2001). Genome of the bacterium Streptococcus pneumoniae
strain R6. J. Bacteriol. 183, 5709–5717. doi: 10.1128/JB.183.19.5709-5717.
2001
Iannelli, F., Pearce, B. J., and Pozzi, G. (1999). The type 2 capsule locus of
Streptococcus pneumoniae. J. Bacteriol. 81, 2652–2654.
Iannelli, F., Santagati, M., Docquier, J. D., Cassone, M., Oggioni, M. R., Rossolini,
G. M., et al. (2004). “Type M resistance to macrolides in streptococci is not due
to the mef(A) Gene, but to mat(A) encoding an ATP-dependent efflux pump,” in
Proceedings of the Forty fourth Interscience Conference on Antimicrobial Agents
and Chemoterapy, Washington, DC.
Iannelli, F., and Pozzi, G. (2004). Method for introducing specific and unmarked
mutations into the chromosome of Streptococcus pneumoniae. Mol. Biotechnol.
26, 81–86. doi: 10.1385/MB:26:1:81
Iannelli, F., and Pozzi, G. (2007). “Protocol for conjugal transfer of genetic elements
in Streptococcus pneumoniae,” in Molecular Biology of Streptococci, eds R.
Hakenbeck and G. S. Chhatwal (Norfolk: Horizon Scientific Press), 525–529.
Iannelli, F., Santagati, M., Santoro, F., Oggioni, M. R., Stefani, S., and Pozzi, G.
(2014a). Nucleotide sequence of conjugative prophage Phi1207.3 (formerly
Tn1207.3) carrying the mef(A)/msr(D) genes for efflux resistance to macrolides
in Streptococcus pyogenes. Front. Microbiol. 5:687. doi: 10.3389/fmicb.2014.
00687
Iannelli, F., Santoro, F., Oggioni, M. R., and Pozzi, G. (2014b). Nucleotide
sequence analysis of integrative conjugative element Tn5253 of Streptococcus
pneumoniae. Antimicrob. Agents Chemother. 58, 1235–1239. doi: 10.1128/AAC.
01764-13
Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., and Sternberg, M. J. (2015). The
phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc.
10, 845–858. doi: 10.1038/nprot.2015.053
Frontiers in Microbiology | www.frontiersin.org
ABC-Cassette Responsible for Macrolide Efflux
Klaassen, C. H., and Mouton, J. W. (2005). Molecular detection of the macrolide
efflux gene: to discriminate or not to discriminate between mef(A) and mef(E).
Antimicrob. Agents Chemother. 49, 1271–1278. doi: 10.1128/AAC.49.4.1271-
1278.2005
Lanie, J. A., Ng, W. L., Kazmierczak, K. M., Andrzejewski, T. M., Davidsen, T. M.,
Wayne, K. J., et al. (2007). Genome sequence of avery’s virulent serotype 2 strain
D39 of Streptococcus pneumoniae and comparison with that of unencapsulated
laboratory strain R6. J. Bacteriol. 189, 38–51. doi: 10.1128/JB.01148-06
Leclercq, R., and Courvalin, P. (1991). Bacterial resistance to macrolides,
lincosamide, and streptogramin antibiotics by target modification. Antimicrob.
Agents Chemother. 35, 1267–1272. doi: 10.1128/AAC.35.7.1267
Lenart, J., Vimberg, V., Vesela, L., Janata, J., and Balikova Novotna, G. (2015).
Detailed mutational analysis of vga(A) interdomain linker: implication for
antibiotic resistance specificity and mechanism. Antimicrob. Agents Chemother.
59, 1360–1364. doi: 10.1128/AAC.04468-14
Martin, B., Prudhomme, M., Alloing, G., Granadel, C., and Claverys, J. P. (2000).
Cross-regulation of competence pheromone production and export in the early
control of transformation in Streptococcus pneumoniae. Mol. Microbiol. 38,
867–878. doi: 10.1046/j.1365-2958.2000.02187.x
Pearce, B. J., Iannelli, F., and Pozzi, G. (2002). Construction of new unencapsulated
(rough) strains of Streptococcus pneumoniae. Res. Microbiol. 153, 243–247.
doi: 10.1016/S0923-2508(02)01312-8
Pozzi, G., Iannelli, F., Oggioni, M. R., Santagati, M., and Stefani, S. (2004). Genetic
elements carrying macrolide efflux genes in streptococci. Curr. Drug Targets
Infect. Disord. 4, 203–206. doi: 10.2174/1568005043340641
Reinert, R. R., Ringelstein, A., van der Linden, M., Cil, M. Y., Al-Lahham, A.,
and Schmitz, F. J. (2006). Molecular epidemiology of macrolide-resistant
Streptococcus pneumoniae isolates in Europe. J. Clin. Microbiol. 43, 1294–1300.
doi: 10.1128/JCM.43.3.1294-1300.2005
Roberts, M. C., Sutcliffe, J., Courvalin, P., Jensen, L. B., Rood, J., and
Seppala, H. (1999). Nomenclature for macrolide and macrolide -lincosamde-
streptogramine B resistance determinants. Antimicrob. Agents Chemother. 43,
2823–2830.
Roberts, M. C. (2008). Update on macrolide-lincosamide-streptogramin, ketolide,
and oxazolidinone resistance genes. FEMS Microbiol. Lett. 282, 147–159.
doi: 10.1111/j.1574-6968.2008.01145.x
Rudolph, K., Bulkow, L., Bruce, M., Zulz, T., Reasonover, A., and Harker-Jones, M.,
et al. (2013). Molecular resistance mechanisms of macrolide-resistant invasive
Streptococcus pneumoniae isolates from Alaska, 1986 to 2010. Antimicrob.
Agents Chemother. 57, 5415–5422. doi: 10.1128/AAC.00319-13
Salles, C., Creancier, L., Claverys, J. P., and Mejean, V. (1992). The high level
streptomycin resistance gene from Streptococcus pneumoniae is a homologue of
the ribosomal protein S12 gene from Escherichia coli. Nucleic Acids Res. 20:6103.
doi: 10.1093/nar/20.22.6103
Santagati, M., Iannelli, F., Cascone, C., Campanile, F., Oggioni, M. R., Stefani, S.,
et al. (2003). The novel conjugative transposon Tn1207.3 carries the macrolide
efflux gene mef(A) in Streptococcus pyogenes. Microb. Drug Resist. 9, 243–247.
doi: 10.1089/107662903322286445
Santagati, M., Iannelli, F., Oggioni, M. R., Stefani, S., and Pozzi, G. (2000).
Characterization of a genetic element carrying the macrolide efflux gene mef(A)
in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 44, 2585–2587.
doi: 10.1128/AAC.44.9.2585-2587.2000
Santagati, M., Lupo, A., Scillato, M., Di Martino, A., and Stefani, S. (2009). Conjugal
mobilization of the mega element carrying mef(E) from Streptococcus salivarius
to Streptococcus pneumoniae. FEMS Microbiol. Lett. 290, 79–84. doi: 10.1111/j.
1574-6968.2008.01408.x
Santoro, F., Oggioni, M. R., Pozzi, G., and Iannelli, F. (2010). Nucleotide sequence
and functional analysis of the tet(M)-carrying conjugative transposon Tn5251
of Streptococcus pneumoniae. FEMS Microbiol. Lett. 308, 150–158. doi: 10.1111/
j.1574-6968.2010.02002.x
Scott, J. R., and Fischetti, V. A. (1983). Expression of Streptococcal M protein in
Escherichia coli. Science 221, 758–760. doi: 10.1126/science.6192499
Seppala, H., Skurnik, M., Soini, H., Roberts, M. C., and Huovinen, P. (1998).
A novel erythromycin resistance methylase gene (ermTR) in Streptococcus
pyogenes. Antimicrob. Agents Chemother. 42, 257–262.
Sharkey, L. K., Edwards, T. A., and O’Neill, A. J. (2016). ABC-F Proteins
mediate antibiotic resistance through ribosomal protection. mBio 7:e01975.
doi: 10.1128/mBio.01975-15
8 July 2018 | Volume 9 | Article 1670
Iannelli et al.
Smith, M. D., and Guild, W. R. (1979). A plasmid in Streptococcus pneumoniae.
J. Bacteriol. 137, 735–739.
Sutcliffe, J., Tait-Kamradt, A., and Wondrack, L. (1996). Streptococcus pneumoniae
and Streptococcus pyogenes resistant to macrolides but sensitive to clindamycin:
a common resistance pattern mediated by an efflux system. Antimicrob. Agents
Chemother. 40, 1817–1824.
Tait-Kamradt, A., Clancy, J., Cronan, M., Dib-Hajj, F., Wondrack, L., Yuan, W.,
et al. (1997). mefE is necessary for erythromycin-resistant M phenotype in
Streptococcus pneumoniae. Antimicrob. Agents Chemother. 41, 2251–2255.
Tatsuno, I., Isaka, M., Masuno, K., Hata, N., Matsumoto, M., and Hasegawa, T.
(2018). Functional predominance of msr(D), which is more effective as mef(A)-
associated than mef(E)-associated, over mef(A)/mef(E) in macrolide resistance
in Streptococcus pyogenes. Microb. Drug Resist. doi: 10.1089/mdr.2017.0277
[Epub ahead of print].
Vitali, L. A., Di Luca, M. C., Prenna, M., and Petrelli, D. (2016). Correlation
between genetic features of the mef(A)-msr(D) locus and erythromycin
resistance in Streptococcus pyogenes. Diagn. Microbiol. Infect. Dis. 84, 57–62.
doi: 10.1016/j.diagmicrobio.2015.08.007
Frontiers in Microbiology | www.frontiersin.org
ABC-Cassette Responsible for Macrolide Efflux
Weisblum, B. (1995). Erythromycin resistance by ribosome modification.
Antimicrob. Agents Chemother. 39, 577–585. doi: 10.1128/AAC.39.3.577
Zhang, Y., Tatsuno, I., Okada, R., Hata, N., Matsumoto, M., Isaka, M., et al.
(2016). Predominant role of msr(D) over mef(A) in macrolide resistance
in Streptococcus pyogenes. Microbiology 162, 46–52. doi: 10.1099/mic.0.
000206
Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2018 Iannelli, Santoro, Santagati, Docquier, Lazzeri, Pastore, Cassone,
Oggioni, Rossolini, Stefani and Pozzi. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is permitted, provided the original
author(s) and the copyright owner(s) are credited and that the original publication
in this journal is cited, in accordance with accepted academic practice. No use,
distribution or reproduction is permitted which does not comply with these terms.
9 July 2018 | Volume 9 | Article 1670